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( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the <t>ORC2</t> <t>protein</t> and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

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1) Product Images from "DNA replication in primary hepatocytes without the six-subunit ORC"

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

Journal: eLife

doi: 10.7554/eLife.102915

Figure Legend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Techniques Used: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Figure Legend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Techniques Used: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Figure Legend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Techniques Used: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Image Search Results

a , Purified human MCM loading proteins analysed by SDS-PAGE and Coomassie staining. Full-length proteins (left, FL) and truncated proteins (right, ΔN): ORC1ΔN, CDC6ΔN, CDT1ΔN. b , Outline of the nuclease footprinting assay. MCM loading reactions are treated with Benzonase followed by quenching with EDTA, SDS and proteinase K. Then DNA is purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation, resolved on a TBE polyacrylamide gel, and stained with SYBR Gold. c , Timecourse of yeast (top) and human (bottom) MCM loading reactions. d , Protein requirements for the DH footprint with truncated human proteins. e , Requirements of MCM loading with full-length proteins. f , Side-by-side comparison of the ORC6 dependency with full-length and truncated proteins. g , As f, testing conditions in which either ORC1, CDC6, or CDT1 was truncated and other proteins full-length. h , As f, with full-length ORC1 and truncated CDC6 and CDT1. i , Effect of geminin on full-length MCM loading reactions. CDT1 and geminin were pre-mixed before reactions were started. j , Salt stability of the DH. MCM was loaded for 30 minutes and then incubated in buffers containing the indicated concentrations of sodium chloride for 15 minutes, followed by dilution and Benzonase treatment.

Journal: bioRxiv

Article Title: MCM Double Hexamer Loading Visualised with Human Proteins

doi: 10.1101/2024.04.10.588848

Figure Lengend Snippet: a , Purified human MCM loading proteins analysed by SDS-PAGE and Coomassie staining. Full-length proteins (left, FL) and truncated proteins (right, ΔN): ORC1ΔN, CDC6ΔN, CDT1ΔN. b , Outline of the nuclease footprinting assay. MCM loading reactions are treated with Benzonase followed by quenching with EDTA, SDS and proteinase K. Then DNA is purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation, resolved on a TBE polyacrylamide gel, and stained with SYBR Gold. c , Timecourse of yeast (top) and human (bottom) MCM loading reactions. d , Protein requirements for the DH footprint with truncated human proteins. e , Requirements of MCM loading with full-length proteins. f , Side-by-side comparison of the ORC6 dependency with full-length and truncated proteins. g , As f, testing conditions in which either ORC1, CDC6, or CDT1 was truncated and other proteins full-length. h , As f, with full-length ORC1 and truncated CDC6 and CDT1. i , Effect of geminin on full-length MCM loading reactions. CDT1 and geminin were pre-mixed before reactions were started. j , Salt stability of the DH. MCM was loaded for 30 minutes and then incubated in buffers containing the indicated concentrations of sodium chloride for 15 minutes, followed by dilution and Benzonase treatment.

Article Snippet: The coding sequences of human ORC1, ORC2, ORC3, ORC4 and ORC5 were codon-optimised for S. frugiperda , synthesised (GeneArt, Thermo Fisher Scientific) and subcloned into modified pBIG1 vectors that contain a pLIB derived polyhedrin expression cassette.

Techniques: Purification, SDS Page, Staining, Footprinting, Extraction, Ethanol Precipitation, Comparison, Incubation

a , Surface rendering and cut-through view of a hSH loaded onto duplex DNA. The C-terminal ATPase site is shown with nucleotides bound at subunit interfaces. b , A superposition of PS1 pore loops and DNA extracted from hDH and hSH show the same configuration. c , MCM hexamer dimerisation changes the path of duplex DNA leading to untwisting and opening of the double helix. d , Surface rendering of the hMO* structure. e , Surface rendering of the yMO structure highlights a different configuration of the ORC complex compared to hMO*. ORC6 is bridges N-terminal MCM and ORC1-5 in both structures. f , A steric clash obtained by modelling the pre-insertion yOCCM (PDB entry 6WGG) demonstrates that hMO* cannot support recruitment of a second MCM, unlike what was observed for the yMO complex. g , Truncated and full length ORC1, CDC6 and CDT1 yield the same hMO* complex, as observed by negative stain 2D averaging.

Journal: bioRxiv

Article Title: MCM Double Hexamer Loading Visualised with Human Proteins

doi: 10.1101/2024.04.10.588848

Figure Lengend Snippet: a , Surface rendering and cut-through view of a hSH loaded onto duplex DNA. The C-terminal ATPase site is shown with nucleotides bound at subunit interfaces. b , A superposition of PS1 pore loops and DNA extracted from hDH and hSH show the same configuration. c , MCM hexamer dimerisation changes the path of duplex DNA leading to untwisting and opening of the double helix. d , Surface rendering of the hMO* structure. e , Surface rendering of the yMO structure highlights a different configuration of the ORC complex compared to hMO*. ORC6 is bridges N-terminal MCM and ORC1-5 in both structures. f , A steric clash obtained by modelling the pre-insertion yOCCM (PDB entry 6WGG) demonstrates that hMO* cannot support recruitment of a second MCM, unlike what was observed for the yMO complex. g , Truncated and full length ORC1, CDC6 and CDT1 yield the same hMO* complex, as observed by negative stain 2D averaging.

Techniques: Staining

a , MCM can be loaded via an ORC6 independent pathway, via two inverted hOCCM complexes that load two hSHs in a process that requires ATP hydrolysis. Free diffusion along duplex DNA would then lead to hDH formation. b, In a variation of the same mechanism, two hOCCMs assembled around duplex DNA are free to diffuse and form a dOCCM. ATP hydrolysis then promotes hDH formation. c, MCM loading with full-length ORC1 can occur in an ORC6 dependent manner and might go through the hMO* intermediate. hMO* might recruit a hSH through an ORC6 interaction with N-terminal MCM. Following hSH release, the same ORC could recruit a second hSH via C-terminal MCM interaction (hOCCM intermediate), resulting in two N-terminally facing hSHs that can assemble a hDH. d, Alternatively, a first hSH could be loaded via OCCM. ORC6 would then interact with the N-terminal domain of hSH forming hMO*. A structural change would then occur, which causes hMO* to transition to a yMO-like state, where hORC is competent for the recruitment of a second hSH, eventually leading to hDH formation. We have however not observed a yMO-like complex formed with human proteins.

Journal: bioRxiv

Article Title: MCM Double Hexamer Loading Visualised with Human Proteins

doi: 10.1101/2024.04.10.588848

Figure Lengend Snippet: a , MCM can be loaded via an ORC6 independent pathway, via two inverted hOCCM complexes that load two hSHs in a process that requires ATP hydrolysis. Free diffusion along duplex DNA would then lead to hDH formation. b, In a variation of the same mechanism, two hOCCMs assembled around duplex DNA are free to diffuse and form a dOCCM. ATP hydrolysis then promotes hDH formation. c, MCM loading with full-length ORC1 can occur in an ORC6 dependent manner and might go through the hMO* intermediate. hMO* might recruit a hSH through an ORC6 interaction with N-terminal MCM. Following hSH release, the same ORC could recruit a second hSH via C-terminal MCM interaction (hOCCM intermediate), resulting in two N-terminally facing hSHs that can assemble a hDH. d, Alternatively, a first hSH could be loaded via OCCM. ORC6 would then interact with the N-terminal domain of hSH forming hMO*. A structural change would then occur, which causes hMO* to transition to a yMO-like state, where hORC is competent for the recruitment of a second hSH, eventually leading to hDH formation. We have however not observed a yMO-like complex formed with human proteins.

Techniques: Diffusion-based Assay

ETV4 transcriptionally controls key genes involved in DNA replication of NSCLC cells. A) Functional analysis showing the top KEGG pathways that are significantly associated with down‐regulated genes in H1299, H1703, and H358T si‐ETV4 groups normalized to negative control (NC) siRNA groups by Human Microarray ( GSE137445 ). B) Clustering analysis showing the gene signature is enriched in the DNA replication pathway. C) IGV tracks showing the enrichment of ETV4 at the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 from ChIP‐seq signals of A549‐shETV4 cells transfected with Flag‐ETV4 plasmids. D) ChIP‐PCR analysis for endogenous or exogenous ETV4 binding to the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 genes in H1299 cells using anti‐ETV4 antibody or H358‐ETV4 cells using anti‐Flag antibody. E) Schematic depicting the core motif of ETV4 binding with its target genes, the wild‐type luciferase reporter constructs, and the mutations in the putative ETV4‐binding sites (mut‐1 or mut‐2 type) of MCMs/ORC1 promoter region. F–J) The wild‐type luciferase reporter plasmids of MCMs (ORC1) were co‐transfected along with ETV4, ETV4‐DBD deletion plasmid, or the empty vector into HEK293T cells. The constructed luc‐MCMs (ORC1)‐mut‐1 or ‐mut‐2 type plasmids were co‐transfected along with ETV4 plasmid or the empty vector into HEK293T cells. Relative luciferase activity was normalized against Renilla luciferase activity, respectively (mean ± SD; n = 4; ordinary one‐way ANOVA with Tukey's multiple comparisons test). **** p < 0.0001; ### p < 0.001, #### p < 0.0001. K) RT‐qPCR analysis of MCM2, ‐4, ‐5, ‐10, and ORC1 mRNA expression in H1299 cells transfected with ETV4 or NC siRNA, Transcript levels were normalized to ACTB gene expression (mean ± SD, n = 3; two‐tailed unpaired t ‐test). ** p < 0.01; *** p < 0.001; **** p < 0.0001. L) Immunoblots showing MCM2, ‐4, ‐5, ‐10, and ORC1 protein levels in NC and ETV4‐knockdown H1299 cells. M,N) Immunoblots showing the Chromatin‐bound proteins (Chrom.) and unbound proteins (Sol.) levels of MCM2, ‐4, ‐5, ‐10, and ORC1 protein in control and sh‐ETV4 A549 cells, or control and ETV4‐overexpression H358 cells.

Journal: Advanced Science

Article Title: Topoisomerase I Inhibition in ETV4‐overexpressed Non‐Small Cell Lung Cancer Promotes Replication and Transcription Mediated R‐Loop Accumulation and DNA Damage

doi: 10.1002/advs.202409307

Figure Lengend Snippet: ETV4 transcriptionally controls key genes involved in DNA replication of NSCLC cells. A) Functional analysis showing the top KEGG pathways that are significantly associated with down‐regulated genes in H1299, H1703, and H358T si‐ETV4 groups normalized to negative control (NC) siRNA groups by Human Microarray ( GSE137445 ). B) Clustering analysis showing the gene signature is enriched in the DNA replication pathway. C) IGV tracks showing the enrichment of ETV4 at the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 from ChIP‐seq signals of A549‐shETV4 cells transfected with Flag‐ETV4 plasmids. D) ChIP‐PCR analysis for endogenous or exogenous ETV4 binding to the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 genes in H1299 cells using anti‐ETV4 antibody or H358‐ETV4 cells using anti‐Flag antibody. E) Schematic depicting the core motif of ETV4 binding with its target genes, the wild‐type luciferase reporter constructs, and the mutations in the putative ETV4‐binding sites (mut‐1 or mut‐2 type) of MCMs/ORC1 promoter region. F–J) The wild‐type luciferase reporter plasmids of MCMs (ORC1) were co‐transfected along with ETV4, ETV4‐DBD deletion plasmid, or the empty vector into HEK293T cells. The constructed luc‐MCMs (ORC1)‐mut‐1 or ‐mut‐2 type plasmids were co‐transfected along with ETV4 plasmid or the empty vector into HEK293T cells. Relative luciferase activity was normalized against Renilla luciferase activity, respectively (mean ± SD; n = 4; ordinary one‐way ANOVA with Tukey's multiple comparisons test). **** p < 0.0001; ### p < 0.001, #### p < 0.0001. K) RT‐qPCR analysis of MCM2, ‐4, ‐5, ‐10, and ORC1 mRNA expression in H1299 cells transfected with ETV4 or NC siRNA, Transcript levels were normalized to ACTB gene expression (mean ± SD, n = 3; two‐tailed unpaired t ‐test). ** p < 0.01; *** p < 0.001; **** p < 0.0001. L) Immunoblots showing MCM2, ‐4, ‐5, ‐10, and ORC1 protein levels in NC and ETV4‐knockdown H1299 cells. M,N) Immunoblots showing the Chromatin‐bound proteins (Chrom.) and unbound proteins (Sol.) levels of MCM2, ‐4, ‐5, ‐10, and ORC1 protein in control and sh‐ETV4 A549 cells, or control and ETV4‐overexpression H358 cells.

Article Snippet: Blots were blocked with 5% nonfat milk in Tris‐Buffered Saline and Tween 20 (TBST), and incubated with antibodies specific for ETV4 (10684‐1‐AP, Proteintech), MCM2 (3619, CST), MCM3 (15597‐1‐AP, Proteintech), MCM4 (13043‐1‐AP, Proteintech), MCM5 (11703‐1‐AP, Proteintech), MCM6 (13347‐2‐AP, Proteintech), MCM7 (11225‐1‐AP, Proteintech), MCM10 (12251‐1‐AP, Proteintech), ORC1 (NBP100‐121, Novus), ORC2 (12739‐1‐AP, Proteintech), ORC3 (sc‐374231, Santa Cruz), ORC4 (13026‐1‐AP, Proteintech), ORC5 (11542‐1‐AP, Proteintech), ORC6 (17784‐1‐AP, Proteintech), Histone‐H3 (17168‐1‐AP, Proteintech), PCNA (13110, CST), phospho‐MCM2 S40 (ab133243, Abcam), CDC45 (15678‐1‐AP, Proteintech), SUPT16H (28598‐1‐AP, Proteintech), SSRP1 (15696‐1‐AP, Proteintech), γ‐H2AX (ET‐1602, HUABio), Flag (14793, CST), His (66005‐1‐Ig, Proteintech), HA (sc‐7392, Santa Cruz), GST (66001‐2‐lg, Proteintech), GAPDH (60004‐1‐Ig, Proteintech) and β‐actin (66009‐1‐Ig, Proteintech) was used as a loading control for Western blot.

Techniques: Functional Assay, Negative Control, Microarray, ChIP-sequencing, Transfection, Binding Assay, Luciferase, Construct, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Expressing, Gene Expression, Two Tailed Test, Western Blot, Knockdown, Control, Over Expression

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Article Snippet: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Origin recognition complex (ORC) mRNA and protein levels in lung adenocarcinoma (LUAD) based on the UALCAN and HPA databases. (A) ORC1‐6 levels in LUAD compared with normal lung samples based on the UALCAN database and (B) expression of ORC protein in LUAD compared with normal lung samples based on the HPA database. *** p < 0.001.

Journal: Cancer Medicine

Article Title: Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance

doi: 10.1002/cam4.5238

Figure Lengend Snippet: Origin recognition complex (ORC) mRNA and protein levels in lung adenocarcinoma (LUAD) based on the UALCAN and HPA databases. (A) ORC1‐6 levels in LUAD compared with normal lung samples based on the UALCAN database and (B) expression of ORC protein in LUAD compared with normal lung samples based on the HPA database. *** p < 0.001.

Article Snippet: The sections were washed three times with phosphate‐buffered saline (PBS) followed by incubation overnight with anti‐ORC1 antibody (Abcam, ab251776 at 1/1500 dilution), ORC2 antibody (Abcam, ab99277 at 1/1000 dilution), ORC3 antibody (Abcam, ab179936 at 1/200 dilution), ORC4 antibody (Abcam, ab235514 at 1/100 dilution), ORC5 antibody (ProteinTech Group, cat no. 11542‐1‐AP, at 1/400 dilution), or ORC6 antibody (Abcam, ab153993 at 1/500 dilution) at 4°C.

Techniques: Expressing

Origin recognition complex (ORC) protein expression in lung adenocarcinoma (LUAD) by immunohistochemistry staining. (A) The level of ORC in LUAD compared with normal lung samples and (B) prognostic values of the ORC levels in LUAD patients.

Journal: Cancer Medicine

Article Title: Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance

doi: 10.1002/cam4.5238

Figure Lengend Snippet: Origin recognition complex (ORC) protein expression in lung adenocarcinoma (LUAD) by immunohistochemistry staining. (A) The level of ORC in LUAD compared with normal lung samples and (B) prognostic values of the ORC levels in LUAD patients.

Techniques: Expressing, Immunohistochemistry, Staining

The correlation between pathological parameters and ORC5

Journal: Cancer Medicine

Article Title: Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance

doi: 10.1002/cam4.5238

Figure Lengend Snippet: The correlation between pathological parameters and ORC5

Techniques: Expressing, Diagnostic Assay

Journal: Cancer Medicine

Article Title: Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance

doi: 10.1002/cam4.5238

Figure Lengend Snippet: The methylation of ORCs in LUAD

Techniques: Methylation

Immune infiltration of the origin recognition complex (ORC) in lung adenocarcinoma. (A) ORC expression in each immune cell type and cell type‐level differential expression analysis results and (B) cancer purity and immune infiltration.

Journal: Cancer Medicine

Article Title: Systemic analysis of the DNA replication regulator origin recognition complex in lung adenocarcinomas identifies prognostic and expression significance

doi: 10.1002/cam4.5238

Figure Lengend Snippet: Immune infiltration of the origin recognition complex (ORC) in lung adenocarcinoma. (A) ORC expression in each immune cell type and cell type‐level differential expression analysis results and (B) cancer purity and immune infiltration.

Techniques: Expressing, Quantitative Proteomics

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